Expression and secretion in Aspergillus nidulans and Aspergillus niger of a cell surface glycoprotein from the cattle tick, Boophilus microplus, by using the fungal amdS promoter system.
A cell surface glycoprotein (Bm86) from cells of the digestive tract of the cattle tick Boophilus microplus, which has been shown to elicit a protective immunological response in vaccinated cattle, was expressed and secreted in the filamentous fungi Aspergillus nidulans and Aspergillus niger by using the fungal amdS promoter system. The cloned gene coded for the Bm86 secretory signal and all of the Bm86 mature polypeptide except for the hydrophobic carboxy-terminal segment. High levels of Bm86 mRNA were detected in the transformed cells. Bm86 polypeptide was secreted from the cells in a soluble form and it was glycosylated, probably to a similar extent to the native glycoprotein. The recombinant product had an apparent molecular mass of 83 to 87 kilodaltons, whereas that predicted from the amino acid sequence was 69 kilodaltons. The Bm86 was expressed at levels of up to 1.8 mg/liter, or approximately 6% of secreted protein under the growth conditions used. No intracellular Bm86 was detected. A general relationship was observed between transformants containing a high number of copies of the expression plasmid and high expression levels.
Turnbull
I F
IF
Department of Genetics, University of Melbourne, Parkville, Victoria, Australia.
Smith
D R
DR
Sharp
P J
PJ
Cobon
G S
GS
Hynes
M J
MJ
D000818:Animals; D001230:Aspergillus / Q000235:genetics*; D001233:Aspergillus nidulans / Q000235:genetics; D001234:Aspergillus niger; D015870:Gene Expression; D005822:Genetic Vectors; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics; D013987:Ticks / Q000235:genetics*; D014170:Transformation, Genetic
D008562:Membrane Glycoproteins; D012333:RNA, Messenger